Our understanding of the Escherichia coli chemotaxis system has progressed during the last 30 or so years. We understand the biochemistry of the binding of the ligands to the transmembrane receptors, the path of phosphate through the two-component signaling system, and the behavior of the flagellar motor in response to the signal. To gain further insights into this complex signal transduction system, the questions of how the receptors generate the initial signal, how this signal is modulated, and how the signal is received by the flagellar motor will be addressed in the future. This proposal focuses on the modulation of the intracellular signal and the interface of this signal with the flagellar motor. We have taken the approach of studying the higher order structure of various protein complexes in order to gain insight into mechanisms of transmission of the chemotaxis signal and of transcriptional regulation. We will focus on the following Specific Aims 1) We will determine how activated CheY binds to the flagellar switch component, FliM. We have found that by forming a complex of a mutant CheY and FliM, we have been able to obtain soluble FliM for the first time after many years of effort. This and other mutant CheY/FliM complexes will be used for NMR and crystallography. 2) We will determine the role the CheAs/CheZ interaction in chemotaxis. A 42 amino acid fragment of the amino terminal end of CheAs ('P1) has been shown to bind and activate CheZ in vitro. The fibronectin display peptides that bind 'P1 will be used in vivo to inactivate CheAs functions without affecting CheAL functions. 3) We will use our technique of isolating stable CheAL/CheA*s/ CheW complexes to test the possibility that CheAs can bind CheW in a phosphorylating complex and then switch to bind CheZ after phosphorylation. 4) We will determine a cognate partner of FlhD for regulation of FlhD dependent-FIhC independent promoters. Once this is established, we will solve the crystal structures of the FlhDC/DNA complex and FlhD/X/DNA complex to determine how complex formation alters DNA binding specificity.